The present invention pertains in general to methods for controlling incorporation of amino acids in expressed recombinant proteins and analogs produced thereby. In particular, the present application pertains to methods for control of norleucine incorporation into recombinant proteins and to analogs of the recombinant proteins produced thereby.
Norleucine may be utilized as a substrate for E. coli methionyl tRNA synthetase, although its K.sub.m is 200-fold higher than that for methionine [Lemoine et al., Eur. J. Biochem., 4, 213-221 (1968)]. Norleucine is a methionine antagonist in many microorganisms [Adelberg, J. Bacteriol., 76, 326 (1958), Hobson, J. Gen. Microbiol., 82, 425-429 (1974), and Lawrence, J. Bacteriol., 109, 8-11 (1972)] and may be synthesized in Serratia marcessens [Kisumi et al., J. Biochem., 80, 333-339 (1976)]. Exogenous radioactively-labelled norleucine may be incorporated into casein proteins when fed to cows [Black et al., J. Am. Chem. Soc., 77, 6082-6083 (1955)]. However, the endogenous occurrence of norleucine in microbial proteins, in particular in recombinant DNA-derived proteins has never been reported.
Kisumi et al. found accumulation of norleucine in a norleucine-resistant mutant of Serratia marcessens [Kisumi et al., Appl. Envir. Microbiol., 34, 135-138 (1977)]. Kisumi et al., J. Biochem., 80, 333-339 (1976) postulated that in Serratia marcesens, norleucine was synthesized via a leucine biosynthetic pathway in which .alpha.-ketobutyrate, .alpha.-ketovalerate, and .alpha.-ketocaproate are substrates in the sequential steps of enzymatic synthesis including a step catalyzed by .alpha.-isopropylmalate synthetase and other enzymes such as isopropyl malate dehydratose, or isopropyl malate dehydrogenase. The final step of leucine synthesis in Serratia marcessens is catalyzed by leucine transaminase. To synthesize these linear .alpha.-ketoacids, acetyl CoA is used in the chain elongation process in the microorganism [Metzler, Biochemistry, "The Chemical Reactions of Living Cells", Academic Press, New York, 1977], but at the present time, it is not known what caused the unusual linear chain elongation reported for these .alpha.-ketoacids.
Wunsch et al., U.S. Pat. No. 3,978,035 describes a method of synthetically producing L-norleucine-13-motilin. The method of Wunsch et al. is based on the replacement of the L-methionine residue located in the 13-position by an L-norleucine residue. This complex chemical synthesis uses the N, N'-dicyclohexylcarbodiimide-N-hydroxysuccinimide method to connect fragments I to VI. A masked polypeptide containing the amino acid sequence is split off from the protective groups with trifluoroacetic acid and then the trifluoracetate and bromide ions are removed.
Rubin, U.S. Pat. No. 4,568,640 describes a method of substituting one amino acid for another in a protein chain to improve selected properties of the protein. The tRNA is modified to carry the substituting amino acid. Modification of the selected tRNA is by misacylation which is facilitated by dimethylsulfoxide, cacodylate and methanol.